IN SILICO ANALYSIS of FUNCTIONAL Snps of ALOX12 GENE and IDENTIFICATION of PHARMACOLOGICALLY SIGNIFICANT FLAVONOIDS AS

Tulasidharan Suja Saranya et al. Int. Res. J. Pharm. 2014, 5 (6) INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230 – 8407

Research Article IN SILICO ANALYSIS OF FUNCTIONAL SNPs OF ALOX12 GENE AND IDENTIFICATION OF PHARMACOLOGICALLY SIGNIFICANT FLAVONOIDS AS LIPOXYGENASE INHIBITORS Tulasidharan Suja Saranya, K.S. Silvipriya, Manakadan Asha Asokan* Department of Pharmaceutical Chemistry, Amrita School of Pharmacy, Amrita Viswa Vidyapeetham University, AIMS Health Sciences Campus, Kochi, Kerala, India *Corresponding Author Email: [email protected]

Article Received on: 20/04/14 Revised on: 08/05/14 Approved for publication: 22/06/14

DOI: 10.7897/2230-8407.0506103

ABSTRACT Cancer is a disease affecting any part of the body and in comparison with normal cells there is an elevated level of lipoxygenase enzyme in different cancer cells. Thus generation of lipoxygenase enzyme inhibitors have suggested being valuable. Individual variation was identified by the functional effects of Single Nucleotide Polymorphisms (SNPs). 696 SNPs were identified from the ALOX12 gene, out of which 73 were in the coding non-synonymous region, from which 8 were found to be damaging. In silico analysis was performed to determine naturally occurring flavonoids such as isoflavones having the basic 3- phenylchromen-4-one skeleton for the pharmacological activity, like Genistein, Diadzein, Irilone, Orobol and Pseudobaptigenin. O-methylated isoflavones such as Biochanin, Calycosin, Formononetin, Glycitein, Irigenin, 5-O-Methylgenistein, Pratensein, Prunetin, ψ-Tectorigenin, Retusin and Tectorigenine were also used for the study. Other natural products like Aesculetin, a coumarin derivative; flavones such as ajoene and baicalein were also used for the comparative study of these natural compounds along with acteoside and nordihydroguaiaretic acid (antioxidants) and active inhibitors like Diethylcarbamazine, Zileuton and Azelastine as standard for the computational analysis. The protein-ligand interaction was studied and the docking analysis was performed by Patch Dock server using the 3D3L human arachidonate 12-lipoxygenase protein. Out of the 19 herbal constituents selected, it was found that Diadzein, Biochanin, Glycitein, Irigenin, 5-O-Methylgenistein, ψ-Tectorigenin and Tectorigenin showed significant binding affinity in inhibiting lipooxygenase enzyme as compared with standard compounds of acteoside, nordihydroguaiaretic acid, Diethylcarbamazine, Zileuton and azelastine. Naturally occurring compounds on further advancement could act as potent lipoxygenase inhibitors. Keywords: Cancer, Lipoxygenase enzyme, SNP, In silico analysis, Docking, Drug likeness

INTRODUCTION partition coefficient) and Total Polar Surface Area (TPSA) Computer aided Drug designing is currently playing a major was calculated by the sum of fragment based contributions role in the field of research and drug discovery. The and correction factors4. TPSA is observed to be an conventional method of development of molecules by indispensable descriptor influencing the drug absorption, synthesis is preceded by the use of drug designing techniques CaCO2 permeability and blood-brain barrier penetration. computationally. Various imperative steps are involved in Molecular Volume calculation is predicted based on cluster drug designing such as structure based and ligand based drug contributions and are fully optimized by the semi empirical designing. In silico approaches based on ligand drug AM1 technique. Rule of 5 is a set of molecular descriptors designing have been performed for the lead molecule used by Lipinski in formulating the Rule of 5. The rule states development. The necessary molecular characteristics of the that most "drug-like" compounds have log P < = 5, molecular ligand have been generated computationally. The various weight < = 500, number of hydrogen bond acceptors < = 10 interactions between the proteins and the ligand are analyzed and number of hydrogen bond donors < = 55. Molecules by docking simulation with the protein target 3D3L1,2. The which do not pursue more than one of these rules may have binding is measured in terms of the score based on the problems with bioavailability. Topological parameter used for protein-ligand interactions. The study of other parameters measuring molecular flexibility is the Number of Rotatable such as detection of cavities was also carried out. Single Bonds (nrotb)6. Rotatable bond is defined as any single non- nucleotide polymorphism plays an essential role in ring bond, bounded to non-terminal heavy (non-hydrogen) identification of individual variations and determination of atom. The rule elucidates the molecular properties like mutations. Drug likeness is a parameter that helps to absorption, distribution, metabolism and excretion (ADME). characterize compounds based on the varied molecular Lipoxygenase (LOXs EC1.13.11) are iron containing properties like hydrophobic character, electron distribution, enzymes which are classified as arachidonate 5-lipoxygenase hydrogen bond formation, size and adaptability of the and 12-lipoxygenase in humans7. Lipoxygenase enzyme may molecule in conjunction with the presence of various initiate the synthesis of a signaling molecule or be involved pharmacophores. The lead molecule affects the in inducing structural or metabolic changes in the cell8. bioavailability, protein binding property, absorption Human lipoxygenases are the enzymes participating in the mechanism, toxicity and stability of the compound3. Mol metabolism of the polyunsaturated fatty acids and catalyzing inspiration was accustomed to calculate parameters like their oxidation to a variety of eicosanoids acting as the relative molar mass, log P, number of hydrogen bond donors secondary signal transducers playing a major role in human or acceptors which are essential to eliminate non-drug like homeostasis9. Phosphorylation via Mitogen activated protein molecules. Sophisticated Bayesian statistics was employed in kinase-2 is critical in controlling and activation of this Mol inspiration for the calculation of the physicochemical enzyme10,11. LOX are implicated in the pathogenesis of properties of the lead molecules. Log P (octanol/water diseases like asthma12, ulcerative colitis13, psoriasis14,

Page 502 Tulasidharan Suja Saranya et al. Int. Res. J. Pharm. 2014, 5 (6) atherosclerosis15 and cancer16. In comparison with normal Aesculetin, ajoene, baicalein, along with the standard drugs tissues, significantly higher concentration of LOX like acteoside, nordihydroguaiaretic acid, intermediates have been found in breast, colon, lung, skin and diethylcarbamazine, Zileuton and azelastine were generated prostate cancers, along with persons with both acute and using Marvin Sketch and saved in the PDB format28. The chronic leukaemias17. Flavonoids are naturally occurring various properties of the ligands were studied using Mol plant metabolite with numerous pharmacological activities. inspiration. The ligands and proteins were subjected to The focus of the research was to verify certain selected energy minimization and finally the docking analysis was flavonoids as lipoxygenase enzyme inhibitor18,19. Certain performed using the Patch Dock server29. Patch Dock is an isoflavones like Genistein, Diadzein, Irilone, Orobol and algorithm for molecular docking. The input for Patch Dock is Pseudobaptigenin; O-methylated isoflavones such as molecules of any type like proteins, DNA, peptides or drugs. Biochanin, Calycosin, Formononetin, Glycitein, Irigenin, 5- The output is a list of potential complexes sorted by shape O-Methylgenistein, Pratensein, Prunetin, ψ-Tectorigenin, complementarily criteria30. The corresponding gene ALOX12 Retusin and Tectorigenin were selected for the study. The of the protein 3D3L has been explored to identify the isoflavones have the basic 3-phenylchromen-4-one skeleton functional effect of Single Nucleotide Polymorphism (SNPs) for the pharmacological activity. Other natural products like retrieved from dbSNP database31 and the deleterious amino Aesculetin, a coumarin derivative; ajoene and baicalein, acid substitution prediction by SIFT32. 696 SNPs have been flavone; were also used for the analysis. Acteoside and recognized in the ALOX12 gene, out of which 73 were in the nordihydroguaiaretic acid denoted as antioxidants and active coding non-synonymous region, 47 in the mRNA UTR inhibitors like diethylcarbamazine, Zileuton and azelastine region, 36 in the coding synonymous region and remaining were used as standard for the comparative computational 540 SNPs in the intron region of the gene. analysis. RESULTS MATERIALS AND METHODS The number of amino acids of the protein was found to be The X-ray crystallographic structure of lipoxygenase domain 541 with the molecular weight as 61089.2 g. The theoretical of human arachidonate 12-lipoxygenase, 12S-type (PDB ID pI was found to be 6.35 with the total number of negatively 3D3L) protein was obtained from RCSB protein data bank20 charged residues (Asp + Glu) as 58 and the total number of at a resolution of 2.60 Å. Water molecules, ligands and other positively charged residues (Arg + Lys) as 52. The total hetero atoms were removed from the protein molecule. number of atoms was found to be 8547. The Extinction Addition of hydrogen atoms to the protein was performed coefficient was 87735 at Abs 0.1 % (= 1 g/l) 1.436, assuming using CHARMm force field. The protein molecules were all pairs of Cys residues form cystines and Ext. coefficient at subjected to stability studies computationally. The primary 86860 with Abs 0.1 % (= 1 g/l) 1.422, assuming all Cys and secondary structure of proteins was studied21,22. residues are reduced. The estimated half-life was 30 hours Parameters studied were Extinction coefficients, half-life, (mammalian reticulocytes, in vitro), > 20 hours (yeast, in aliphatic index and Grand average of hydropathicity vivo) and > 10 hours (Escherichia coli, in vivo). The (GRAVY)23. The Extinction coefficients indicate how much instability index (II) was computed to be 48.66. The Aliphatic light a protein absorbs at a certain wavelength. They are index was found to be 86.21. Grand average of measured in units of M-1 cm-1, at 280 nm in water. The half- hydropathicity (GRAVY) was computed to be -0.259. The life is the prediction of time it takes for half of the amount of target protein secondary structure analysis was performed. protein in a cell to disappear after its synthesis in the cell. The The Alpha helix (Hh) was found to be 226 (41.77 %), 310 N-terminal of the sequence considered is M (Met). Prot helix (Gg), Pi helix (Ii), Beta bridge (Bb) and Bend region Param is based on the N-end rule which correlates the half- (Ss) was 0 %. The Extended strand (Ee) was 56 (10.35 %), life of a protein to the identity of its N-terminal residue24. The Beta turn (Tt) was found to be 29 (5.36 %) and Random coil aliphatic index of a protein is defined as the relative volume (Cc) 230 as 42.51 %. The LOX enzyme was found in large occupied by aliphatic side chains (alanine, valine, isoleucine, quantity in cytoplasm. The ligand molecules were subjected and leucine). It helps to influence the thermo stability of to molecular property calculation using Mol inspiration. The globular proteins. The aliphatic index of a protein is based on properties calculated were total polar surface area (TPSA), the following formula25 mentioned in Eq. 1. number of atoms, molecular weight, number of hydrogen bond acceptors and number of hydrogen bond donors, Aliphatic index = X (Ala) + a *X (Val) + b *X (Ile) + X (Leu) nviolations and number of rotatable bonds (Table 1). The Eq. 1 calculation of drug likeness and bioactivity scores of ligands Where X (Ala), X (Val), X (Ile), and X (Leu) are mole percent (100 X mole fraction) of alanine, valine, isoleucine, and leucine were studied using the parameters GPCR ligands, ion channel modulators, kinase inhibitors, nuclear receptor ligands, The coefficients a and b are the relative volume of valine side protease inhibitors and other enzyme targets (Table 2). After chain (a = 2.9) and of Leu/Ile side chains (b = 3.9) to the side the preliminary analysis, the protein molecule was subjected chain of alanine. The GRAVY value for a peptide or protein to the energy minimization. The minimized protein and is calculated as the sum of hydropathy values of all the amino ligands were saved in PDB format. The Accelrys Discovery 33 acids, divided by the number of residues in the sequence. studio 3.5 visualizer was used for visualization of the PDB Detection of the location of the enzyme was performed format of the protein 3D3L as shown in Figure 1. Docking computationally using WoLFPSORT26,27. WoLFPSORT analysis was performed by Patch Dock server and the energy predicts the sub cellular localization sites of proteins based values were computed as in Table 3 and the column chart of on their amino acid sequences. The ligand structures of the Patch Dock score was represented in Figure 2. In silico naturally occurring flavonoids such as Genistein, Diadzein, analysis was also carried out to determine the damaging Irilone, Orobol, Pseudobaptigenin, Biochanin, Calycosin, Single Nucleotide Polymorphisms (SNPs) which might be Formononetin, Glycitein, Irigenin, 5-O-Methylgenistein, responsible for the variations in individuals. 696 SNPs were Pratensein, Prunetin, ψ-Tectorigenin, Retusin, Tectorigenin, analyzed using the gene ALOX12 of the corresponding Page 503 Tulasidharan Suja Saranya et al. Int. Res. J. Pharm. 2014, 5 (6) protein 3D3L. It was found that 73 were in the coding non- intron region of the gene. 8 SNPs were found to be damaging synonymous region, 47 in the mRNA UTR region, 36 in the in nature with deleterious amino acid substitutions which was coding synonymous region and remaining 540 SNPs in the displayed in Table 4 with its score values.

Table 1: Drug likeness calculation of the ligand molecules

Compounds mi Log P TPSA natoms MW nON nOHNH nviolations nrotb volume Diadzein 2.56 70.67 19 254.24 4 2 0 1 216 Genistein 2.268 90.895 20 270.24 5 3 0 1 224.1 Irilone 2.637 89.135 22 298.25 6 2 0 1 239.9 Orobol 1.778 111.12 21 286.239 6 4 0 1 232.1 Pseudobaptigenin 2.928 68.907 21 282.251 5 1 0 1 231.9 Biochanin 2.804 79.901 21 284.267 5 2 0 2 241.6 Calycosin 2.377 79.901 21 284.267 5 2 0 2 241.6 Formononetin 3.095 59.673 20 268.268 4 1 0 2 233.6 Glycitein 2.377 79.901 21 284.267 5 2 0 2 241.6 Irigenin 2.086 118.6 26 360.318 8 3 0 4 300.7 5-O-Methylgenistein 2.544 79.901 21 284.267 5 2 0 2 241.6 Pratensein 2.086 100.13 22 300.266 6 3 0 2 249.6 Prunetin 2.804 79.901 21 284.267 5 2 0 2 241.6 ψ-Tectorigenin 2.283 100.13 22 300.266 6 3 0 2 249.6 Retusin 2.835 79.901 21 284.267 5 2 0 2 241.6 Tectorigenin 2.283 100.13 22 300.266 6 3 0 2 249.6 Zileuton 2.465 66.56 16 236.296 4 3 0 2 203.2 Aesculetin 1.021 70.667 13 178.143 4 2 0 0 144.6 Ajoene 1.802 17.071 13 234.411 1 0 0 8 207.9 Baicalein 2.682 90.895 20 270.24 5 3 0 1 224.1 Diethylcarbamazine 0.881 26.785 14 199.298 4 0 0 2 209.6 Nordihydroguaiaretic 3.476 80.912 22 302.37 4 4 0 5 287.9 acid azelastine 4.817 38.135 27 381.907 4 0 0 3 349.3 acteoside -0.449 245.29 44 624.592 15 9 3 11 532.5

TPSA: total polar surface area; natoms: number of atoms; MW: molecular weight; nON: number of hydrogen bond acceptors; nOHNH: number of hydrogen bond donors; nrotb: number of rotatable bonds

Table 2: Calculation of Bioactivity scores of ligand molecule

Compounds GPCR Ion channel Kinase Nuclear receptor Protease Enzyme ligand modulator inhibitor ligand inhibitor inhibitor genistein -0.22 -0.54 -0.06 0.23 -0.68 0.13 Diadzein -0.31 -0.064 -0.2 0.04 -0.83 0.02 Irilone -0.2 -0.67 -0.16 0.07 -0.7 0.02 Orobol -0.17 -0.5 0.03 0.28 -0.63 0.15 Pseudobaptigenin -0.21 -0.66 -0.13 0.01 -0.73 0.02 Biochanin -0.23 -0.59 -0.06 0.23 -0.66 0.07 Calycosin -0.25 -0.65 -0.08 0.06 -0.78 0.01 Formononetin -0.3 -0.69 -0.19 0.05 -0.8 -0.02 Glycitein -0.24 -0.66 -0.08 0.07 -0.77 0.01 Irigenin -0.21 -0.57 0 0.02 -0.6 0.03 5-O-Methylgenistein -0.21 -0.58 -0.06 0.19 -0.67 0.04 Pratensein -0.19 -0.56 0.02 0.22 -0.65 0.09 Prunetin -0.23 -0.59 -0.06 0.23 -0.66 0.07 ψ-Tectorigenin -0.23 -0.4 -0.06 0.12 -0.65 0.11 Retusin -0.29 -0.65 -0.1 0.03 -0.75 0.04 Tectorigenin -0.22 -0.64 -0.01 0.1 -0.72 0.05 Zileuton 0.01 -0.14 -0.28 -0.56 0.15 0.74 aesculetin -1.05 -0.61 -1.06 -0.81 -1.17 -0.22 ajoene -0.67 -0.99 -1.32 -0.74 -0.63 0.24 baicalein -0.12 -0.18 0.19 0.17 -0.35 0.26 diethylcarbamazine -0.23 -0.13 -0.34 -0.76 -0.44 -0.18 nordihydroguaiaretic acid 0.03 0.11 -0.05 0.14 0.01 0.13 azelastine 0.12 -0.18 0.13 -0.56 -0.18 0.18 acteoside 0 -0.54 -0.31 -0.24 0.06 0

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Table 3: Docking analysis result using Patch Dock server

Compounds Patch dock genistein 4190 Diadzein 5416 Irilone 4758 Orobol 4468 Pseudobaptigenin 4560 Biochanin 4832 Calycosin 4654 Formononetin 4598 Glycitein 4938 Irigenin 5472 5-O-Methylgenistein 5472 Pratensein 4634 Prunetin 4704 ψ-Tectorigenin 5076 Retusin 4614 Tectorigenin 4870 Zileuton 2610 aesculetin 3076 ajoene 4022 bacalein 1196 diethylcarbamazine 3908 nordihydroguaiaretic acid 4966 azelastine 5556 acteoside 7368

Table 4: In silico analysis of Single Nucleotide Polymorphisms (SNPs)

S. No SNP Amino acid change Score Median 1 rs41359946 T38N 0.04 2.77 2 rs114985038 D134H 0.05 2.78 3 rs140116643 A497V 0.05 2.78 4 rs141346813 R348 -1 -1 5 rs143493293 T364I 0.03 2.77 6 rs147000782 E40K 0.01 2.9 7 rs147158964 R404Q 0.03 2.77 8 rs148602792 K133N 0.01 2.78

Figure 1: Minimized protein 3D3L prepared for docking analysis

Figure 2: In silico analysis of natural compounds as Lipoxygensae inhibitors

Page 505 Tulasidharan Suja Saranya et al. Int. Res. J. Pharm. 2014, 5 (6)

DISCUSSION ALOX12 gene and 3D3L protein were investigated in this The preliminary in silico analysis was performed to check the work by evaluating the influence of functional SNPs through parameters such as the primary and secondary structure computational analysis. Of a total of 696 SNPs analyzed, it analysis of the protein 3D3L. Primary analysis involved the was found that 73 were in the coding non-synonymous calculation of Extinction coefficient, estimated half-life, region, 47 in the mRNA UTR region, 36 in the coding Instability index, Aliphatic index and Grand average of synonymous region and remaining 540 SNPs were in the hydropathicity (GRAVY) values. The instability index (II) intron region of the gene. During the analysis, 8 SNPs have was computed to be 48.66 and this classifies the protein as been predicted as damaging and 22 SNPs as tolerant in being in an unstable state. The Aliphatic index was found to nature. The SNPs which were damaging could be modified to be 86.21 and therefore the protein demonstrates considerable prevent the individual variations. The lipoxygenase enzyme thermo stability. GRAVY value of -0.259 indicates that the inhibitor can prove to be effective in case of individuals with protein was hydrophilic in nature. Secondary structure cancer. Naturally occurring compounds on further analysis showed considerable stability of the protein. The advancement could act as potent lipoxygenase inhibitors. LOX enzyme was found in large quantity in cytoplasm. The Further investigations are essential for the development of ligand molecules were generated using Marvin Sketch and potent lipoxygenase inhibitor chemical entities for the saved in the PDB format. They were subjected to molecular prevention and treatment of cancer. property calculation using Mol inspiration. The properties calculated were total polar surface area (TPSA), number of REFERENCES 1. René T, Mikael HC. 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